Method to treat collagenous connective tissue for implant remodeled by host cells into living tissue

ABSTRACT

The invention relates to a method of treatment of collagenous connective tissue removed from a donor for implant into a recipient which is re-habited or re-colonized by host cells without an immune rejection and inflammatory reaction. After removal from the donor the tissue is trimmed and thereafter soaked in a cold stabilizing solution having a temperature range of 4 to 10 degrees centigrade. The tissue is then soaked at a predetermined temperature in a polyglycol, salt, hydrogen peroxide, and phosphate buffer first solution of predetermined quantities and concentrations and of sufficient ionic strength to permit ground substances to dissociate such that the collagen fibers remain stable. The tissue is then soaked in an alcohol and water solution at a predetermined temperature for a sufficient period of time to remove the residue of the first solution. Following the removal of the residue, the tissue is soaked at a predetermined temperature in a third solution of an anti-inflammatory agent, an anti-thrombic agent, alcohol, and water or sequentially in an anti-inflammatory agent, alcohol, and water solution, and then in an anti-thrombic agent, alcohol and water solution and thereafter stored.

This Application is a Divisional Application of Prior application Ser.No. 10/235,017, filed Sep. 23, 2002.

FIELD OF THE INVENTION

The present invention relates to a process for treating collagenousconnective tissue from an animal donor for implant in an animal or humanrecipient.

BACKGROUND OF THE INVENTION

The present invention relates to a process for treating collagenousconnective tissue or structural support tissue removed from an animaldonor for implant into a recipient without an immune and inflammatoryrejection. Collagenous connective tissue or structural support tissuemay be heart valve tissue, blood vessel, pericardium, omentum, fascia,tendon, ligament, intestine, cartilage, bone, membrane, or other suchtissue.

Implants from animal donors into a human recipient to correct defectivebody components are known in the prior art. For instance, hemostaticcollagen implants and collagen injections have been used for hemostasisand tissue augmentation; homograft and xenograft tissue heart valveshave been widely implanted with variable results. The implant materialsfor these procedures were derived from animal origins that containedcollagen, elastin, pericardium, and cells along with the proteins andother substances within the extracellular matrices. In the prior art thetreatment of the implant depended upon its intended use. Implantmaterials generally were either intended for temporary implant or forpermanent incorporation by the recipient. As an example of a temporaryimplant, hemostatic collagen may be used for emergencies to arrest bloodloss; once hemostasis occurs, there is no further utility for thecollagen. The concern where the implant is temporary is that majorcomplications such as infection, pyrogenic shock or a major foreign bodyreaction by the recipient might occur. Another example of a temporaryimplant is the absorbent suture which is intended for temporary use soas to permit a wound to heal as the tissue regains its strength. Wherethe temporary implant material is derived from an animal, the implantmaterial need not be homogeneous; it can be denatured and slightly toxicto the recipient.

Implant materials intended for permanent or long lasting implantationmay provide structural support for a body part or may be an activefunctional organ such as a kidney, liver, or heart. For activefunctional organs, an immune compatible organ is most desirable.Preferably, it is harvested from a donor whose tissue closely matchesthat of the recipient.

In the case of permanent structural support, implants derived frombiological sources are treated in the prior art before implantationutilizing one of three basic strategies.

The first basic strategy involves chemical modification, i.e., chemicalmodification of functional groups of tissue components, crosslinking,surface modification, etc. It can also increase the mechanical strengthas well as the durability of the tissue by adding crosslinking to tissuefibers. Chemical modification can reduce the antigenicity and alterother properties of the tissue when it is transplanted into a differentspecies. One of the most successful modifications of connective tissuefor use as a bioprothesis is the glutaraldehyde fixation or crosslinkingof porcine heart valves and bovine pericardium. However, there arelimitations on this technology. Glutaraldehyde treated tissue has beenshown to be toxic and host cells will not infiltrate tissue subjected tothis treatment. The physical properties of glutaraldehyde treated tissueare very different from the physical properties of native untreatedtissues. In the case of glutaraldehyde treated tissue for a heart valveprosthesis, calcification is known to be a major cause of valve failure.Numerous methods have been developed to modify the physical propertiesof glutaraldehyde treated tissue to overcome these limitations. Thesemethods include but are not limited to the following:

a) treatment with detergent or surfactant after glutaraldehydecrosslinking;

b) covalently binding diphosphonates to the glutaraldehyde treatedtissue;

c) covalently binding amino-substituted aliphatic functional acid to theglutaraldehyde treated tissue;

d) covalently binding sulfated polysaccharides, especially chondroitinsulfate after glutaraldehyde crosslinking;

e) treatment with ferric or stannic salts either before of afterglutaraldehyde crosslinking;

f) incorporation of polymers, especially elastomeric polymers, into theglutaraldehyde treated tissue; or

g) immersing glutaraldehyde treated tissues in solutions of awater-soluable phosphate ester or a quaternary ammonium salt or asulfated higher aliphatic alcohol.

None of these methods, however, has been entirely successful.

The second basic strategy for long-term implantation involves making apiece of tissue with no vital cells. Some examples of this are thefollowing:

a) Decellularization (killing cells by hypotonic shock then followedwith nucleases). Goldstein, U.S. Pat. Nos. 5,613,982; 5,632,778;5,899,936 and 5,843,182;

b) Controlled autolysis Jaffe, U.S. Pat. Nos. 5,843,180; 5,843,181 and5,720,777;

c) Killing of cells by radiation, as shown in Schinstein, U.S. Pat. Nos.5,795,790; 8,843,431; 5,843, 717 and 5,935,849; and Badylak, U.S. Pat.No. 6,126,686); peracetic acid (Badylak U.S. Pat. No. 6,126,686);

d) Acid treatment (Abraham, U.S. Pat. No. 5,993,833); and

e) Other processes in the prior art construct composites of purifiedcollagenous material with a synthetic scaffold (Bell, U.S. Pat. No.6,051,750). The objective of all of these methods is to create a matrixthat the host can accept such that cells in the recipient's body canmigrate into the matrix and eventually remodel the material into aliving tissue.

The third strategy involves the preservation or incorporation of livingcells in the transplated material. Tissues removed from the donors havea finite time limit before cells in the tissues die due to autolysis asa result of lack of oxygen. Conditions such as lowering the temperatureand placing the tissues in solutions containing ischemic protectionagents can keep cells vital for an extended time. Cryopreservation hasbeen used to preserve the vitality of cells in tissues to an even longertime. However, the number of cells that survive in a host issubstantially affected by the time lapse between harvesting andcryopreservation, and by the length of time the tissue has beenpreserved, by the thawing process and the implant procedures. Thesurviving cells will thereafter face a very hostile environment in thehost unless there is a close genetic match or there is initiated animmune suppressive therapy.

The treatment of scaffold material to form living tissue outside thehost has become a popular research topic. In an incubation environment,in order to have enough cells to infiltrate and grow in the scaffoldmaterial, the cells must be able to or stimulated to divide and growrapidly.

To transition from a growing to a stable phase, such rapidly dividingand growing cells must be controlled. Once the cells are controlled,whether they will remain controlled after implant in the host is a majorconcern under this third strategy. The basic limitation that pervadesthis third strategy of the prior art is the lack of a sterilizationmethod that can preserve living mammalian cells.

While the third strategy either keeps the cells from the donor alive orutilizes facilities outside of the recipient's body to culture orincubate living cells into the implants, by contrast the second strategyutilizes the recipient's cells as the donor source for cells and therecipient's body environment as an incubator. The current inventionprovides a process where implant materials will interact with therecipient's body without an immune and inflammatory rejection and thebody cells will migrate to the implant to remodel the implant intoliving tissue.

Immune rejection is a major problem in transplantology. The major causeof immune rejection is the difference between the cell surface moleculesof the donor and the recipient. If the implant contains donor livingcells, this problem is prolonged and amplified as long as the donorcells are living and proliferating. If the implanted tissue does notcontain living cells, the dead cells still present a problem. For thisreason, methods have been developed to extract the remnants of deadcells from tissue through the use of detergents, solvents, etc. Suchmethods require extraction to be very aggressive to ensure that all theunwanted materials are extracted. Under such harsh conditions, tissuematrix components which are critical to the integrity of the tissue arealso destroyed. The destruction of the tissue matrix components can besubtle and difficult to detect and often not easily observed using lightmicroscopy or electron microscopy.

It has been suggested in the prior art that polyethylene glycol (PEG)may reduce immune response of recipients to allogeneic transplants(“Heart Preservation Solution Containing Polyethylene Glycol: AnImmunosuppressive Effect” by Collins, et al. in Lancet, 338:390 (1991)).In one study a 30% reduction has been observed in the incidence of acuterejection in a group of heart transplant recipients in which the donororgan had been stored at 4° C. in a solution containing 5% PEG. In asubsequent study, PEG produced a modest but statistically significantincrease in rat liver allograft survival time from 9.6 to 11.9 days (see“The Immunosuppressive Effect of Polyethyene Glycol in a Flush Solutionfor Rat Liver Transplantation” by Tokunaga, et al. in Transplantation,54:756-8 (1992)). In these studies, the transplant organ was merelysoaked in the PEG solution without subsequent cryopreservation. In U.S.Pat. No. 4,938,961, Collins, et al., discloses an organ preservationsolution containing polyethylene glycol, along with a variety of furtheringredients including: 30-40 mM NaOH, 100 mM lactobionic acid, 25 mMKH₂PO₄, 10 mM KOH, 30 mM raffinose, and 3 mM glutathione. This solutionis used for the transport of an organ from a donor to a recipient. U.S.Pat. No. 6,280,925 to Bruckbank discloses a tissue pretreatment solutioncontaining PEG and glutathione for use prior to cryopreservation. In thecurrent invention, by incubating PEG with tissues under a high ionicstrength condition, the collagen molecules remain insoluable but theinteraction between proteoglycans and collagens is weakened. In thismanner, proteoglycans on the surface of collagen fibers are replaced byPEG while preventing the collagen fibers from collapsing andaggregating.

While it is important to remove and mask substances antigenic to therecipients, it is also important to consider other factors relevant tothe behavior and survival of the implanted materials. Acute and chronicinflammation are major defense mechanisms our bodies use against foreigninvading materials as well as removing damaged materials resulting frominjury or cell death. The mechanisms are initiated by inflammatory cellsarriving at the site of foreign material. They digest the foreignmaterial by oxidation. The digested material or the digestion processfurther recruits more inflammatory cells. As a result, the cyclecontinues until all the foreign or damaged “wounded self” materials areremoved. Very often, an acute inflammatory reaction can cause damagedtissues to be even more vulnerable to further recruitment ofinflammatory reaction thereby resulting in chronic inflammation. Anexample is chronic arthritis; inflammatory cells continue to attack analready inflamed joint matrice from previous injury or disease. Theinflamed tissues recruit a further inflammatory reaction which damagesthe joint matrice even further and results in a vicious cycle that leadsto a permanent disabling of the joint. In most cases, inflammatoryreactions subside as the injured tissue or foreign bodies are clearedfrom the recipient. In the field of implantable biomedical implants,long term success of an implantable biomaterial depends on the hostprevention of the inflammatory reaction against the implant.

In the present invention, the foreign implant becomes acceptable to thehost or recipient by a process that renders the implant less oxidizableand thus less inflammatory. This is achieved in part by using hydrogenperoxide to oxidize the foreign implant.

Thus, the present invention provides a process for treating collagenousconnective tissue such that the tissue is implanatable without an immuneand inflammatory rejection. The process permits the collagen matrix toremain intact and permits the residual antigenic components to be maskedby polyethylene glycol; the process permits collagen fibers to remainstructurally viable and the tissue to be oxidized to reduce recruitmentof inflammatory cells; the process permits the tissue to incorporatewater insoluable anti-inflammatory agents that inhibit the arrival ofinflammatory cells at the implant site, and permits the tissue fibers toabsorb an anti-thrombosis agent on their surface.

SUMMARY OF THE INVENTION

There is, therefore, provided according to the present invention, amethod for processing collagenous connective tissue from an animal donorsource such that the collagen fibers may be implanted in a recipientwithout an immune and inflammatory rejection.

The present invention is directed to a process for treating collagenousconnective tissue that permits the collagen fibers to remainstructurally intact and permits the tissue to be implanted in therecipient without an immune and inflammatory rejection. After removalfrom the donor, the collagenous connective tissue is trimmed in salineand thereafter the collagen fibers are stabilized in a cold stabilizingsolution having a temperature range of 4 to 10 degrees centigrade. Thecold stabilizing solution may be a saline solution where the collagenfibers are soaked preferably for a period less than 48-hours, or analcohol/water solution where the soaking time preferably does not exceed30 days. After the tissue is stabilized, it is submerged and soaked in asolution comprised of polyglycol, a salt, a phosphate buffer, and anoxidizing agent. The concentration of the polyglycol is in the range of1% to 15% and its molecular weight may be in the range of 2,000 Daltonsto 20,000 Daltons; the salt concentration may be in the range of2.5M-4.5M (moles per liter of solution). The phosphate buffer isselected from the group consisting of sodium phosphate and potassiumphosphate. It is preferable that the buffer have a concentration of0.05M with a pH of 7.4. However, the pH may have a range between 6.5 and7.8 and the concentration may range from 0.02 to 0.1M; and the oxidizingagent preferably is hydrogen peroxide having a concentration in therange of 0.1% to 2%. Ozone may also be used as an oxidizing agent in ananother method in a concentration range of 1-500 ppm, preferably in therange of 20-40 ppm.

Following soaking in the first or masking solution, the tissue is washedin a second solution comprised of alcohol and water where the alcoholmay be selected from the group consisting of ethanol, iso-propanol,n-propanol, and combinations of different alcohols.

After washing the residue remaining on the tissue from soaking in thepolyglycol, salt, phosphate buffer and oxidizing agent solution, thetissue is further soaked in a third solution containing alcohol, waterand an anti-inflammatory agent selected from the group consisting ofindomethacin, ibuprofin, aspirin, choline salicylate, difunisal,magnesium salicylate, magnesium choline salicylate, salsalate,flurbiprofen, fenoprofen, ketoprofen, naprosen, naproxen sodium,oxaprozin, diclofenac sodium, diclofenac misoprostol, etodolac, indocin,ketorolac, natumetone, sulindac, tolmetin, sulfinpyrazone, dipyridamole,ticlopidine, valdecoxib, rofecoxib, piroxicam, meloxicam, meclofenamatesodium, mefenamic, cyclophosphamide, cyclosporine micromulsion,chlorambucil, anagrelide, clopidogrel, and cilostazol, where theconcentration of the anti-inflammatory agent is in the range of 10 to200 mg/liter. Following soaking in the third solution, the tissue isfurther soaked in a solution of alcohol, water and an anti-thrombicagent which may be selected from the group consisting of heparin,ardeparin, enoxaparin, tinzaparin, danapariod, lepiruden and hirudin.The concentration of the anti-thrombic agent may be in the range of 100to 1,000 IU/ml.

In an alternative method, after washing, the tissue may be soaked in asolution of alcohol, water, an anti-inflammation agent, and ananti-thrombic agent having a concentration in the range of 100 to 1,000IU per ml. The anti-inflammatory agent and anti-thrombic agent in thealternative method are selected from the same groups identified above.

In another alternative method, for non-cardiovascular applications suchas orthopedics, neurological, and urological applications, afterwashing, the tissue is soaked in an anti-inflammatory solutioncontaining alcohol, water and an anti-inflammatory agent selected fromthe group consisting of indomethacin, ibuprofin, aspirin, cholinesalicylate, difunisal, magnesium salicylate, magnesium cholinesalicylate, salsalate, flurbiprofen, fenoprofen, ketoprofen, naprosen,naproxen sodium, oxaprozin, diclofenac sodium, diclofenac misoprostol,etodolac, indocin, ketorolac, natumetone, sulindac, tolmetin,sulfinpyrazone, dipyridamole, ticlopidine, valdecoxib, rofecoxib,piroxicam, meloxicam, meclofenamate sodium, mefenamic, cyclophosphamide,cyclosporine micromulsion, chlorambucil, anagrelide, clopidogrel, andcilostazol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating the steps comprising the method forprocessing collagenous connective tissue taken from an animal donor forimplant into a recipient without an immune rejection and an inflammatoryrejection.

DETAILED DESCRIPTION

The major defense mechanism against the implant of foreign tissue oragainst injured and dead host cells is commonly known as acute andchronic inflammation. Inflammation occurs when the recipient's defensemechanism recognizes the foreign tissue or injured or dead host cellsand recruits inflammatory cells such as macrophages andpolymorphonucleocytes. Upon the arrival of inflammatory cells at theinvasive site, the host body secretes inflammatory granules containingenzymes that digest the foreign materials by an oxidation process. Thisdigestion process recruits even more inflammatory cells and as a resultthe cycle continues until all of the foreign materials or damaged cellsare removed. In most cases, the inflammatory reaction subsides as theinjured tissue or invading foreign body is cleared from the recipient;this usually means that the inflammatory site is depleted of the foreignmaterial that had the potential of being further oxidized.

In the field of implantable biomedical implants, long-term success of animplanatable biomaterial depends on the strength of the hostinflammatory reaction. The present invention is directed toward aprocess which renders the implant less oxidizible and thus essentiallyeliminating an inflammatory condition.

A key oxidizing component of the inflammatory cell and the inflammatorygranule is the free radical oxygen molecule in the form of a peroxide.The current invention utilizes hydrogen peroxide as an oxidant which isthe same oxidant naturally generated in a host body during inflammatoryreactions. Hydrogen peroxide is capable of oxidizing a number ofbiological molecules within oxidizible functional groups such asaldehydes, amines, lipids and fats. Because the initial oxidationreaction can cause the denaturation of tissue fibers, resulting indamaged tissue, it is desirable and necessary to protect the tissuefiber while the oxidation reaction is carried out. It has been foundthat collagen molecules and fibers in animal tissue are very stable insalt concentrations above 3 Normal at neutral pH. Under such conditions,collagen is not soluble but if previously solublized, will precipitate.In the present invention, this condition is utilized to stabilize thetissue while the oxidation reaction is carried out.

To render an implant less inflammatory, the present invention utilizesan anti-inflammatory agent which is incorporated into the implant.However, there are a number of anti-inflammatory agents widely used inmedicine that produce unwanted side effects. For instance, steroidalanti-inflammatory drugs such as Glucocorticoids derived from structuresof steroids possess biological characteristics that will result in suchside effects.

Likewise, some non-steroidal agents are undesirable because they vary intheir action and effectiveness. Soluble drugs are also undesirable forincorporation in an implant because they are cleared from the implanttoo quickly. In the present invention anti-inflammatory drugs are chosenwhich are not easily soluble or quickly cleared from the implant. It hasbeen found that when indomethacin or other anti-inflammatory agents aredissolved in 50% ethanol, they will penetrate well-preserved biologicalmatrices. Also, since indomethacin has a very low solubility in water,the infiltrate indomethacin will be cleared from the implanted materialvery slowly.

Connective tissue is composed mainly of collagen, elastin fibers,collagenous proteins, proteoglycans, lipids and nucleic acids, smallsoluble organic molecules, minerals, salts and water. When extractingnon-collagen materials from implant tissue, the small substances caneasily be washed away unless within living cell. In such case, killingthe cell will release the substances to the extracellular space and thusmake them easily removable. However, the larger substances such asproteins, proteoglycans, and nucleic acids are more difficult to remove.A classical way to purify collagen is to subject the collagen to a highconcentration of salt. This causes the collagen to precipitate while theproteoglycans and nucleic acids are soluble. The high salt concentrationalso breaks the interaction of proteoglycans and nucleic acids and theirpropensity to cling to collagen. Thus, soaking tissue in high saltconcentrations removes certain non-collagenous material while keepingthe tissue intact. To facilitate removal of non-collagenous materialinside cells, the tissue may first be treated in ethanol which destroysand extracts the lipid membrane of cells.

The integrity of the collagen molecules and fibers in tissue must beprotected while the non-collagenous material is being extracted and theremaining material is oxidized. Polyethylene glycol (PEG) at aconcentration of 4% has been found to precipitate collagen and can beused in tissue cultures to keep newly sensitized collagen insoluble. Incase of cell cultures, because the cells are attached to the bottom of aplastic dish or a support surface, a large volume of medicum is pouredon top to feed the cells which promotes an environment similar toconditions inside a recipient body. Unlike cells surrounded by a tissuematrix environment in a living organism, most of the collagen made bycells is released into the medium on top of the cells. In order toanalyze the collagen made by the cells and avoid dealing with largevolumes of medium, PEG is added and it has been found that this resultsin all of the collagen remaining at the bottom of the dish. Thus, as thecollagen is shipped outside of the cells, it is kept insoluble by thePEG and cannot float away. Since PEG binds to collagen and otherproteins tightly, it acts naturally as a masking agent of non-extractedantigenic sites in the tissue.

FIG. 1, schematically presents the process of this invention and itsembodiments. Schematics 1 and 2 identify the steps of removal andtrimming of tissue that has been harvested from an animal donor.

After harvesting, the tissue is kept from being degraded by placing thetissue in a physiological saline solution at low temperature, preferably4-10 degrees centigrade for a period not to exceed 48 hours. Blood andother soluble substances are rinsed away using large volumes of coldsaline and the excess tissue is trimmed away. Collagen matrix inphysiological saline with the proper pH and low temperature is stableand proteolytic (enzymatic degradation of collagen matrix) activitiesresulting from cell death can also be minimized under such conditions.

Referring now to schematic 3 of FIG. 1, it is therein indicated that thetissue may be stabilized in an alcohol/water solution for a period notto exceed 30 days at a temperature in the range from 4 to 10 degreescentigrade. Since collagen is not soluble in 50% ethanol, the collagenmolecules cannot unwind from the collagen fibers contained in thetissue. Intact collagen fibers are stable against most degradativeactivities which are also fixed and inactivated in 50% ethanol. Thetissue may be stabilized in a cold stabilizing solution when thesolution is a saline solution; however the soaking period in a salinesolution should preferably not exceed 48 hours but may be extended to 72hours. Where the cold stabilizing solution is an alcohol and watersolution, the soaking period should be for a period less than 30 days.

After the tissue is treated in the stabilizing solution, the tissue isthen transferred into a first solution, as indicated in schematic 4,that is comprised preferably of polyethylene glycol (PEG)₅% (molecularweight 8,000), 0.5-0.6% hydrogen peroxide, 4 M salt, and sodiumphosphate or potassium phosphate at 0.05M, pH 7.4 and incubated orsoaked at a temperature of preferably 4 degrees C. for a period of 96hours. In the preferred method, the ratio of solution to tissue isgreater than 50 cc/2 gm of tissue to ensure that the tissue is submergedin the solution and that the treatment is complete. This could beaccomplished at ratio in the range of 25 cc up to 2 liters.

At concentrations of PEG greater than 15%, host cells will fuse and thusconcentrations above this level are to be avoided. High concentrationsof PEG have been used to fuse mouse myeloma cells and mouse spleen cellsin the production of monoclonal antibodies. Likewise, higherconcentrations of hydrogen peroxide in excess of 2% are to be avoided toprevent excessive oxidation. The preferred concentration of hydrogenperoxide is 0.5 to 0.6%. Hydrogen peroxide in the range of 0.1 to 2% maybe used for the treatment of tissue in the process of this invention. Inanother embodiment the oxidizing agent may be ozone having a preferableconcentration of 20-40 ppm; however, the range of concentration may befrom 1-500 ppm.

The polyglycol identified in schematic step 4 may be polyethyleneglycol, polypropylene glycol or derivatives of these polymers. Thepreferred molecular weight of the polyglycol is 8,000 Daltons; however,the molecular weight of the polyglycol may be in the range of 2,000 to20,000 Daltons. As previously stated, the preferred concentration ofpolyglycol is 5%; the concentration of polyglycol in the solution mayvary however between 1% and 15%. The salt referred to in schematic 4 ispreferably sodium chloride with a concentration between 2.5 to 4.5 M.The salt can be selected from the group consisting of sodium chloride,potassium chloride, sodium bromide, potassium bromide, sodium sulfate,potassium sulfate, ammonium chloride and ammonium sulfate. The phosphatebuffer is selected from the group consisting of sodium phosphate andpotassium phosphate. It is preferable that the buffer have aconcentration of 0.05M with a pH of 7.4. However, the pH may have arange between 6.5 and 7.8 and the concentration may range from 0.02 to0.1M. When soaking the tissue in the first solution containing thepredetermined quantities of polyglycol, oxidizing agent, salt andphosphate buffer as above recited, the solution will be of sufficientionic strength to cause ground substances to dissociate from the tissuewhile the collagen fibers remain intact. The tissue preferably should besoaked in the polyglycol, oxidizing agent, salt and phosphate buffersolution for a period of 96 hours; however, it has been found that thetissue may alternatively be soaked for periods in the range of 12 hoursto 14 days.

After treating the tissue described in schematic 4 above, the tissue isthen washed in an alcohol/water solution as indicated in schematic 5. Inthe washing step set forth in schematic 5, the salt in the tissue iswashed away by 50% ethanol preferably until a negligible amount of saltis left in the tissue. The concentration of alcohol may be between 25%to 70% and the alcohol may be ethanol, iso-propanol, n-proponol orcombination of these different alcohols. It is preferred that thetemperature of the alcohol be at 4 degrees centigrade however thewashing range may be from 4 to 25 degrees centigrade and the time forwashing may be as long as 30 days. It is preferred that the washingoccur overnight or approximately for 16 hours.

After the washing step referred to in schematic 5, the tissue is furthersoaked in a 50% ethanol solution containing 20 mg/liter of indomethacinand 250 IU/ml of heparin. In non-cardiovascular applications (forexample, tendons and ligaments, conduits for nerve guides, tissuemembranes such as tissue patches, urological conduits, etc.) heparin isnot needed and can be omitted. The amount of indomethacin oranti-inflammatory agent should be in the range used for humans on adose/kg body weight basis. High dosages of the anti-inflammatory agentare to be avoided because the tissue can be damaged and become toxic tohost cells.

Referring now to schematics 6, 7, and 8, it can be seen that after thestep of washing the tissue in alcohol/water as stated in schematic 5,the tissue may be treated before storage in any of 3 separate ways: (1)tissue may be sequentially treated as shown in schematic 6 followed byschematic 7; (2) the tissue may be treated as indicated in schematic 6and then sent to storage; (3) or the tissue may be treated as describedin schematic 8 and then sent to storage. The preferred treatment is thetreatment described in schematic 8 where the tissue is treated in asolution containing both anti-inflammatory and anti-thrombotic agents inalcohol/water. Although endomethycin and heparin are the preferredant-inflammatory and anti-thrombotic agents, there are numerous othersuch agents that might be used. For instance, the anti-inflammatoryagent may be an analgesic or anti-pyretic selected from the groupconsisting of indomethacin, ibuprofin, aspirin, choline salicylate,difunisal, magnesium salicylate, magnesium choline salicylate,salsalate, flurbiprofen, fenoprofen, ketoprofen, naprosen, naproxensodium, oxaprozin, diclofenac sodium, diclofenac misoprostol, etodolac,indocin, ketorolac, natumetone, sulindac, tolmetin, sulfinpyrazone,dipyridamole, ticlopidine, valdecoxib, rofecoxib, piroxicam, meloxicam,meclofenamate sodium, mefenamic, cyclophosphamide, cyclosporinemicromulsion, chlorambucil, anagrelide, clopidogrel, and cilostazol; theanti-thrombic agent may be an anti-coagulant selected from the groupconsisting of heparin, ardeparin, and enoxaparin, tinzaparin,danapariod, elpiruden and hirudin.

The concentration of the anti-inflammatory agent in schematics 6, 7 or8, is preferred to be 20 mg per liter; however, the concentration may bein the range of 10 mg to 200 mg per liter. The soaking time preferred inschematics 6, 7, and 8 is preferred to be 24 hours to allow the agent topenetrate the tissue uniformly.

After the treatment of tissue as set forth above in the embodiments ofthe methods of this invention, the tissue may be stored as shown inschematic 9. The methods of storage are well known in the prior art andthe storage medium preferably would be 50% ethanol with the range ofstorage temperature between 0° C. and 6° C. to minimize bioburdeneffects.

The following are examples of tissue treated in accordance with theprocess of the present invention:

EXAMPLE 1

Freshly harvested bovine mesenteric arteries were stored in 50% alcoholfor one week and then treated (or soaked) in a solution containing 5%PEG (molecular weight 8,000D), 0.5% hydrogen peroxide, 4 M NaCl and 0.05M phosphate buffered at pH 7.0 for 96 hours at 4° C. The arteries werewashed in 50% ethanol twice and then further treated (or soaked) in asolution of indomethacin (50 mg/l) and heparin (250 IU/ml) in 50%ethanol overnight. The treated arteries were further soaked in 50%ethanol containing 20% glycerol, 5% PEG (MW=10,000), indomethacin (50mg/l), and heparin (250 IU/ml). The arteries were then freeze-driedusing a FTS Durastop/Duratop lyophilyzer system. The freeze-driedarteries were subjected to hydrogen peroxide sterilization by StarService (Hayward, Calif.) with a Sterrad-100 hydrogen peroxide gas phasesterilizer using a standard validated sterilization program. The tissueswere rinsed in saline for 30 minutes before the following studies weredone.

1. Shrinkage Temperature—treated arteries have a shrinkage temperaturebetween 62-65° C., which is comparable to fresh untreated graft tissues.

2. Enzyme Digestion—treated arteries are similar to fresh untreatedtissue in its ability to resist pepsin digestion. Pepsin digestion oftreated arteries for 24 hours at 4° C. released less than 5% of non-saltprecipitable hydroxyproline indicating that the collagen fibers in thetissue material are intact after treatment.

3. Tensile Strength—mechanical stress-strain analysis was carried out onnine treated arteries and three fresh untreated control arteries. Thetest method was designed and conducted according to ANSI/AAMI VP20-1994Section 8.3.2. The results demonstrate that tensile strength as well asthe longitudinal pull strength of Corograft™-D is similar to untreatedcontrol bovine mesenteric arteries. The results of these testsdemonstrate that the longitudinal tensile strength of treated arteriesis similar to the untreated fresh control bovine mesenteric arteries.The mean and standard deviation for the longitudinal tensile strengthfor the nine treated strengths of 33.2±6.5 Newtons. The mean andstandard deviation for the extension of the nine treated arteries was42.8±4.0 mm beyond the original 50 mm length compared with the controlswhich had a value of 48.9±3.9 mm extension beyond the original 50 mmlength.

4. Compliance—the compliance of treated arteries and untreated controlbovine arterial vessels were studied using an in-house test set-up formeasuring the Dynamic Compliance in accordance with ANSI/AAMI VP20-1994Section 8.10. The set-up consists of the Wieting Cardiac Valve Analyzerwhich provided a pulse rate at 72 beats per minute and physiologicsystolic/diastolic pressure ratios adjustable to 80/40, 120/80, 160/120and 200/160 mm Hg per the FDA Guidance. The change in diameter wasmeasured by means of the SCIMED Coronary Imaging Catheter. The aortic(or input) pressure was measured by means of a Baxter Uniflo PressureTranducer with Honeywell Electronics for Medicine Pressure Amplifier,which is converted to a digital data input into a computer dataacquisition system. The results show that treated arteries has a dynamiccompliance similar to untreated bovine mesenteric arteries. The meansand standard deviation for the compliance for the nine treated arterysamples at a systolic/diastolic pressure ratio of 80/40 mmHg was6.58±3.24 (% radial change per mmHg) compared with the fresh controlwhich had a compliance value of 8.82±1.65 for ten cycles at the samesystolic/diastolic pressure ratio.

5. Suture Retention Strength—suture retention strength of treatedarteries reconstituted in saline and fresh untreated bovine arteries wascarried out in-house using a Chatillon Universal Tension/CompressionTester Model LTCM-6, (Ametek Test and Calibration Instruments, Largo,Fl) and an Omegadyne “S” Beam Load Cell Model LC 101-25. The test methodwas designed and conducted according to ANSI/AAMI VP20-1994 Section 8.8.Prolene 6-0 suture (Ethicon) was used for 90° cut (normally used forproximal anastomosis) and 7-0 was used for 45° oblique cut (normallyused for distal anastomosis to the target coronary artery vessel). Themean suture retention force for nine treated artery samples and threeuntreated control bovine arterial vessels are listed in Table 1 below:TABLE 1 Suture Retention Force (Kg) Toe Heel Right side 45° C. cut(oblique) Treated arteries 0.202 ± 0.037 0.119 ± 0.042 0.199 ± 0.038Untreated control 0.206 ± 0.064 0.223 ± 0.085 0.190 ± 0.028 90° C. cut(straight) Treated arteries 0.247 ± 0.058 0.272 ± 0.024 0.303 ± 0.060Untreated control 0.211 ± 0.051 0.253 ± 0.042 0.248 ± 0.045

6. Suture Hole Elongation—a suture hole elongation study of treatedarteries and untreated control bovine mesenteric arteries was carriedout using the Chatillon Universal Tension/Compression Tester ModelLTCM-6. The study was designed and conducted following the FDA DraftGuidance for the Preparation of Research and Marketing Applications forVascular Graft Protheses (1993) Section 11.1.4.

Five continuous loops were placed 2 mm from the 90° cut end of eachsamples (9 treated arteries and 3 control samples) using Prolene 6-0suture. Initial test using a 5-mg weight at the other end of the sutureloop did not result in any measurable suture hole elongation.

The amount of weight used was determined based on a revision of the FDArecommended equation, WT=5(FL*)/n where FL*=PA or Pn(d/2)² instead ofFL=Pd. Therefore weights were gradually added in subsequent tests from 0to 19 grams. The results are shown in Table 2 below. TABLE 2 Suture HoleElongation at Test sample 9 grams 19 grams Treated arteries 0.04 ± 0.03mm 0.09 ± 0.05 mm Control 0.11 ± 0.03 mm 0.17 ± 0.01 mmThese tests indicate that the suture holes in treated arteries did notelongate as much as those in the control grafts.

7. Burst Strength—the burst strength analysis of treated arteriesreconstituted in saline and untreated control bovine mesenteric arterieswas carried out. The study was designed and conducted according toANSI/AAMI VP20-1994 Section 8.3.3. The test set-up consists of apressure reservoir connected to a compressed air supply providing 0-125psi of pressure via a pressure regulator.

One end of a test sample was connected to the outlet of the reservoirand the other end was connected to a pressure transducer (Omega ModelPX302-100G) where the output was routed to a computer data acquisitionsystem. During the test, water pressure in the reservoir increased at arate of approximately 0.07 psi/second until the test sample burst. Themean and standard deviation of the bursting pressure for the ninetreated artery samples was 14.7±2.6 psi for the three untreated controlbovine mesenteric arteries was 15.2±1.7 psi (greater than seven timesthe mean normal blood pressure of 100 mm Hg in human). The differencebetween the bursting pressure for treated arteries and untreated controlfresh arteries is not statistically significant.

8. Kink Radius—the kink radius of treated arteries reconstituted insaline was studied (according to ANSI/AAMI VP20-1994 Section 8.9) usingan set-up consisting of a series of brass cylinders with diameters of4.5, 5.5, 7.0, 8.0, 9.0, 10.0, 11.5, 12.5 and 13.5 mm. A static pressurereservoir was used to maintain a hydrostatic bovine mesenteric arterysamples show that the average kink radius of treated arteries (5.6±0.8mm) is about 1.75 mm larger than the control untreated samples (3.8±0.3mm)

9. Crush Resistance (Philogenesis Protocol No. 019, Test Report No.TR-008)—Crush resistance test of Corograft™-D reconstituted in salinewas carried out (following the FDA Guidance for the Preparation ofResearch and Marketing Applications for Vascular Graft Prothesis, 1993,Section 11.1.6.). The results shows that Corograft™-D has a slightlygreater crush resistance force (38.7±0.17.4 g) than control untreatedgrafts (26.0±5.7 g) at 2 mm (more than 50%) deflection.

10. Cytotoxicity, MEM elution—treated arteries were evaluated by the MEMelution cell culture cytotoxicity test carried out by Nelson Laboratory,Salt Lake City, Utah in accordance with ANSI/AAMI/ISO 10993-5: 1999,10993-12: 1996, 10993-1: 1997. The results showed that no rounding ofcell or cell lysis (mild reactivity) was observed with the arteryextracts (24 hours at 37° C.

11. Skin Sensitization Test (ISO Magnusson Kligman Method, 2extracts—treated arteries were tested using the Skin Sensitization Test(ISO Magnusson Kligman Method) in guinea pigs. This study wassubcontracted to NAMSA, Irvine, Calif. by Nelson Laboratory, Salt LakeCity, Utah in accordance with ISO 10993-10. Samples were reconstituted(rehydrated) with saline (1.2 g/200 ml PSB) for 5 minutes. Thereconstituted samples were then extracted with saline (1.2 g of tissue/6ml of saline) at 37° C. for 72 hours. Mixtures of extracts, Freund'sComplete Adjuvent and SWFI (1:1:2) were injected intradermally in guineapigs to induce a potential allergic response. Topical applications ofthe extracts at the injection sites (after 7 days) were used as a secondinduction. On day 21 the animals were challenged for measurements of anallergic response at day 22-25. The results showed that no evidence ofthe treated arteries causing delayed dermal contact sensitization in theguinea pig.

12. Skin irrigation Test (ISO intracutaneous reactivity, 2 extracts)—theISO intracutaneous reactivity of the treated arteries was tested inrabbit. This study was subcontracted to NAMSA, Irvine, Calif. by NelsonLaboratory, Salt Lake City, Utah, and Biological Test Center, Irvine,Calif. according to ISO 10993-10. Samples were reconstituted(rehydrated) with saline (1.6 g/200 ml PSB) for 5 minutes. Reconstitutedsamples of extracts (1.6 g of samples extracted with 8 ml of saline orcottonseed oil at 37° C. for 72 hours were injected intracutaneouslyinto the dorsal skin of the rabbits (5 sites with 200 ml injection/sitein each rabbit). The extracts of treated arteries had a negligiblePrimary Irritation Index.

13. Systemic Toxicity—ISO system toxicity (mice, 2 extracts)—potentialsystemic toxicity of the treated arteries was tested on mice. The studywas subcontracted to NAMSA, Irvine, Calif. by Nelson Laboratory, SaltLake City, Utah in accordance with ISO-10993-11. Samples werereconstituted (rehydrated) with saline (1.6 g/200 ml PBS) for 5 minutes.Reconstituted samples of extracts (1.6 g of samples extracted with 8 mlof saline or cottonseed oil at 37° C. for 72 hours) were injected inmice did not cause a decrease in body weight over a period of 72 hoursand no mortality or evidence of systemic toxicity from the extracts wasobserved.

14. Subchronic Toxicity: Intravenous Toxicity withHistopathology—potential subchronic toxicity for the treated arterieswas tested in rats. The study was subcontracted to NAMSA, Irvine, Calif.by Nelson Laboratory, Salt Lake City, Utah (according to ISO 10993-11)using the Intravenous Toxicity with Histopathology test. Samples werereconstituted (rehydrated) with saline 6.9 g/200 ml PBS) for 5 minutes.Reconstituted samples of extracts at 37° C. for 72 hours (6.9 g ofsample was covered with 35 ml of saline for extracts on days 1-7, and7.9 g of sample was covered with 40 ml of saline for extracts on days8-14). Test animals received an injection of 10 ml/kg (body weight) viathe laterial tail vein once each day for 14 consecutive days. There wasno significant evidence of systemic toxicity from the extracts of thetest article injected intravenously into rats.

15. Genotoxicity, Ames Test (Salmonella Typhimurium Reserve MutationAssay—The Ames Test was conducted by Nelson Laboratory, Salt Lake City,Utah in accordance with ANSI/AAMI/ISO 10993-3:1993, 10993-12:1996, onextracts from treated arteries. Test graft samples were reconstituted in200 ml of saline for 5 minutes. The reconstituted samples were thenextracted by 20 ml of saline at 37° C. for 72 hours. The extracts testedagainst the five tester strains did not meet the criteria for apotential mutagen.

16. ASTM Hemolysis Test—the treated arteries were tested by NelsonLaboratory, Salt Lake City, Utah (according to ASTM F756-00, July, 2000)using human blood samples. The results showed a 4.8% corrected hemolyticindex indicating the device is “slightly hemolytic”.

17. Partial Tromboplastin Time Test (PTT Test)—the PTT test wasconducted by Nelson Laboratory, Salt Lake City, Utah in accordance withANSA/AAMI/ISO 10993-4: 1993. Three samples of treated arteries (0.8 g)were incubated in 4.0 ml of human citrated plasma for PTT test. Theresult showed that there is no clot formation during the 10-minute testinterval for any of the 6 replicate samples. This is expected sincetreated arteries is incorporated within heparin.

18. Prothrombin Time Test (PT Test)—the PT test was conducted by NelsonLaboratory, Salt Lake City, Utah according to ANSI/AAMI/ISO10993-4:1993. Four treated artery samples were studied and the resultsindicated that treated arteries did not show a clot during the 10 minutetest interval.

EXAMPLE 2

Freshly harvested bovine pericardium was stored in 50% alcohol for oneweek and then treated in a solution containing 5% PEG, 0.5% hydrogenperoxide, 4 M NaCl and 0.05 M phosphate buffered at pH 7.0 for 48 hoursat 4° C. The pericardial tissues were washed in 50% ethanol twice andthen further treated in a solution of indomethacin (50 mg/l) and heparin(250 IU/ml) in 50% ethanol overnight. The treated pericardial tissueswere further soaked in 50% ethanol containing 20% glycerol, 5% PEG(MW=10,000), indomethacin (50 mg/l), and heparin (250 IU/ml). Thepericardial tissues were then freeze-dried using a FTS Durastop/Duratoplyophilyzer system. The freeze-dried pericardial tissues were subjectedto hydrogen peroxide sterilization by Star Service (Hayward, Calif.)with a Sterrad-100 hydrogen peroxide gas phase sterilizer using astandard validated sterilization program. The tissues were rinsed insaline for 30 minutes before the following study was done:

1. Cell Adhesion and Attachment Study—this study was conducted using astandard cell culture protocol. The study was done by growing smoothmuscle cells, fibroblasts or endothelial cells on treated bovinepericardium. Treated pericardial tissues were used in order to provide aflat surface for cell culture studies. Results demonstrate that thetreated tissues are non-toxic and support the attachment, adherence andproliferation of these different cell types.

2. In a separate study, treated pericardial and control tissues treatedwith gluatarldehyde placed on top of culture dishes with confluentcells. Glutaraldehyde treated pericardial tissue were toxic resulting inmost cells dying around the tissues. However, cells surrounding thetreated pericardial migrated onto the tissue samples.

EXAMPLE 3

Bovine mesenteric arteries and porcine mammary arteries were stored in50% alcohol for one week and then treated in a solution containing 5%PEG, 0.5% hydrogen peroxide, 4 M NaCl and 0.05 M phoshate buffered at pH7.0 for 48 hours at 4° C. The arteries were washed in 50% ethanol twiceand then further treated in a solution of indomethacin (20 mg/l) andheparin (250 IU/ml) in 50% ethanol overnight. The tissues were rinsed insaline for 30 minutes before the following studies were done:

1. Sheep descending aorta to circumflex coronary artery bypass implantstudy—treated porcine mammary arteries, glutaraldehyde treated porcineand bovine conduits as well as PTFE grafts were implanted in 10 sheep asdescending aorta to circumflex artery bypass for up to 202 days. Theresults of this study are summarized in the Table 3 below: TABLE 3Animal Group PO Days* Patency 1 Treated Porcine Artery 202 S Patent 2Treated Porcine Artery 194 S Patent 3 Treated Porcine Artery 162 SPatent 4 Treated Porcine Artery 202 S Patent 5 Treated Porcine Artery189 S Patent 6 Glutaraldehyde Treated Porcine Artery 7 D Obstructed 7Glutaraldehyde Treated Bovine Artery 155 S Obstructed 8 GlutaraldehydeTreated Bovine Artery 16 D Obstructed 9 PTFE 16 D Obstructed 10 PTFE 58D Obstructed*Post-Operative days: S—Sacrificed, D—Death

2. Sheep ascending aorta to circumflex coronary artery bypass implantstudy treated bovine and porcine arteries were implanted in 12 sheep asascending aorta to circumflex artery bypasses. Two sheep were implantedwith autologous azygos veins as controls. The purpose of this series isto compare treated arteries to acceptable controls rather than PTFE orglutaraldehyde treated grafts that are known to fail as coronarybypasses. Thirteen animals survived and were sacrificed between 150-172days. One sheep with a treated artery died at 10 days from a non-graftrelated event. The results are shown in Table 4 below: TABLE 4 PO NoSmall/ Moderate/Lesion Animal Group Days* Patency¹ Lesion/ThrombusLesion/Thrombus² Thrombus³ 3 Autologous Vein 168/S + + 4 Autologous Vein165/S + + 1 Treated Porcine Artery  10/D 2 Treated Porcine Artery172/S + + 8 Treated Porcine Artery 162/S + + 9 Treated Porcine Artery158/S + + 10 Treated Porcine Artery 151/S + + 11 Treated Porcine Artery160/S + + 12 Treated Porcine Artery 155/S + + 5 Treated Bovine Artery162/S + + 6 Treated Bovine Artery 155/S + + 7 Treated Bovine Artery154/S + + 13 Treated Bovine Artery 152/S + + 14 Treated Bovine Artery152/S + +Total number of animals survived = 13*Post-Operative days: S—Sacrificed, D—Early Death¹Patency was confirmed by Angio²Small occlusion with thrombus (<1%)³Moderate occlusion with thrombus (<10%)

EXAMPLE 4

Fresh porcine heart valve leaflets stored in 50% alcohol for two weekswere treated in a solution containing 5% PEG, 0.5% hydrogen peroxide, 4M NaCl and 0.05 M phosphate buffered at pH 7.0 for 48 hours at 4° C. Theheart valve leaflets were washed in 50% ethanol twice and then mountedon stents (Labcor Laboratories, Belo Horizonte, Brazil). The mountedvalves were further treated in a solution of indomethacin (20 mg/l) andheparin (250 IU/ml) in 50% ethanol overnight. The tissues were rinsed insaline for 30 minutes before implanted as mitral heart valvesreplacement in juvenile sheep. These implants were recovered after afive-month implant period and the valves were patent with no signs oftissue over growth or calcification. Valve leaflet tissues were coveredwith lining cells consistent with endothelial cells.

While I have shown and described examples and embodiments of a method oftreatment of collagenous connective tissue for implant into a recipientwithout an immune or inflammatory rejection, it is to be understood thatthe method is subject to many modifications without departing from thescope and spirit of the claims as recited herein.

1. A method for processing collagenous connective tissue having astructure of collagen fibers from an animal donor source such that uponimplant of said tissue into a recipient said tissue is acceptable to therecipient without an immune and inflammatory rejection, comprising thesteps of: (a) stabilizing the collagen fibers contained in saidcollagenous connective tissue in a cold stabilizing solution for aperiod of time and at a temperature sufficient to retain the structureof the collagen fibers; (b) soaking said collagenous connective tissuein a first solution comprising predetermined quantities andconcentrations of polyglycol, an oxidizing agent, salt and phosphatebuffer at a predetermined temperature where said first solution is ofsufficient ionic strength to permit ground substances to dissociate fromsaid collagenous connective tissue such that the collagen fibers remainstable; (c) after step (b), washing said collagenous connective tissuein predetermined quantities and concentrations of a second solutioncomprising alcohol and water for a sufficient period of time at apredetermined temperature to sufficiently remove the residue of saidfirst solution therefrom; (d) soaking said collagenous connective tissuein a third solution comprising a predetermined quantity andconcentration of an anti-inflammatory agent at a predeterminedtemperature; and (e) soaking said collagenous connective tissue in afourth solution comprising a predetermined quantity and concentration ofan anti-thrombic agent at a predetermined temperature.
 2. The methodrecited in claim 1 wherein said polyglycol is selected from the groupconsisting of polyethylene glycol and polypropylene glycol.
 3. Themethod recited in claim 1 wherein said salt is selected from the groupconsisting of sodium chloride, potassium chloride, sodium bromide,potassium bromide, sodium sulfate, potassium sulfate, ammonium chlorideand ammonium sulfate.
 4. The method recited in claim 1 wherein saidoxidizing agent is hydrogen peroxide.
 5. The method recited in claim 1wherein said anti-inflammatory agent is selected from the groupconsisting of indomethacin, ibuprofin, aspirin, choline salicylate,difunisal, magnesium salicylate, magnesium choline salicylate,salsalate, flurbiprofen, fenoprofen, ketoprofen, naprosen, naproxensodium, oxaprozin, diclofenac sodium, diclofenac misoprostol, etodolac,indocin, ketorolac, natumetone, sulindac, tolmetin, sulfinpyrazone,dipyridamole, ticlopidine, valdecoxib, rofecoxib, piroxicam, meloxicam,meclofenamate sodium, mefenamic, cyclophosphamide, cyclosporinemicromulsion, chlorambucil, anagrelide, clopidogrel, and cilostazol. 6.The method recited in claim 1 wherein said anti-inflammatory agent insaid third solution is an analgesic.
 7. The method recited in claim 1wherein said anti-inflammatory agent in said third solution is anantipyretic.
 8. The method recited in claim 1 wherein the temperature ofsaid third solution is in the range of 4 to 25 degrees Centigrade. 9.The method recited in claim 1 wherein said anti-thrombic agent in saidfourth solution is selected from the group consisting of heparin,ardeparin, enoxaparin, tinzaparin, danapariod, lepiruden and hirudin.10. The method recited in claim 1 wherein said anti-thrombic agent is ananticoagulent.
 11. The method recited in claim 1 wherein said alcohol insaid second solution is selected from the group consisting of ethanol,iso-propanol, and n-propanol.
 12. The method recited in claim 1 whereinthe concentration of said alcohol in said second solution is in therange of 25% to 75%.
 13. The method recited in claim 1 wherein theconcentration of said anti-inflammatory agent in said third solution isin the range of 10 to 200 mg/liter.
 14. The method recited in claim 1wherein the concentration of said anti-thrombic agent in said fourthsolution is in the range of 100 to 1000 IU/ml.
 15. The method recited inclaim 1 wherein the concentration of said polyglycol in said firstsolution is in the range of 1% to 15% and the molecular weight of saidpolyglycol is in the range of 2,000 Daltons to 20,000 Daltons.
 16. Themethod recited in claim 1 wherein the concentration of said salt in saidfirst solution is in the range of 2.5M to 4.5M.
 17. The method recitedin claim 1 wherein the concentration of said oxidizing agent in saidfirst solution is in the range of 0.1% to 2%.
 18. The method recited inclaim 1 wherein the temperature of said cold stabilizing solution is inthe range of 4 to 10 degrees Centigrade.
 19. The method recited in claim18 wherein said cold stabilizing solution is a saline solution, furthercomprising soaking said collagenous connective tissue in said salinesolution for a period less than 48 hours.
 20. The method recited inclaim 18 wherein said cold stabilizing solution is an alcohol and watersolution, further comprising soaking said collagenous connective tissuein said alcohol and water solution for a period less than 30 days. 21.The method recited in claim 19 further comprising, after the steprecited in claim 19, placing said collagenous connective tissue in analcohol solution and soaking said collagenous connective tissue thereinfor a period less than 30 days.
 59. The method recited in claim 1wherein said tissue is soaked in said first solution for a period in therange of 12 hours to 14 days.
 62. The method of claim 1 where saidphosphate buffer is selected from the group consisting of sodiumphosphate and potassium phosphate.
 63. The method of claim 1 wherein theconcentration of said phosphate buffer is in the range of 0.02 to 0.1Mwith a pH in the range of 6.5 to 7.8.
 68. The method recited in claim 1further comprising the steps of soaking said collagenous connectivetissue in a 50% ethanol solution containing predetermined quantities andconcentrations of glycerol, polyethylene glycol, indomethacin, andheparin, freeze-drying said collagenous connective tissue with alyophilyzer system, and sterilizing said collagenous connective tissuewith a hydrogen peroxide gas phase sterilizer.
 71. The method recited inclaim 1 further comprising the steps of freeze-drying said collagenousconnective tissue with a lyophilyzer system, and sterilizing saidcollagenous connective tissue with a hydrogen peroxide gas phasesterilizer.
 74. The method recited in claim 1 wherein said oxidizingagent is ozone.
 77. The method recited in claim 74 wherein the range ofconcentration of said oxidizing agent is in the range of 1-500 ppm. 79.The method recited in claim 74 wherein the range of concentration ofsaid oxidizing agent is in the range of 1-500 ppm
 80. Collagenousconnective tissue specimen having a structure of collagen fibers from ananimal donor source prepared in a method having the steps of: (a)stabilizing the collagen fibers contained in said collagenous connectivetissue in a cold stabilizing solution for a period of time and at atemperature sufficient to retain the structure of the collagen fibers;(b) soaking said collagenous connective tissue in a first solutioncomprising predetermined quantities and concentrations of polyglycol, anoxidizing agent, salt and phosphate buffer at a predeterminedtemperature where said first solution is of sufficient ionic strength topermit ground substances to dissociate from said collagenous connectivetissue such that the collagen fibers remain stable; (c) after step (b),washing said collagenous connective tissue in predetermined quantitiesand concentrations of a second solution comprising alcohol and water fora sufficient period of time at a predetermined temperature tosufficiently remove the residue of said first solution therefrom; (d)soaking said collagenous connective tissue in a third solutioncomprising a predetermined quantity and concentration of ananti-inflammatory agent at a predetermined temperature; and (e) soakingsaid collagenous connective tissue in a fourth solution comprising apredetermined quantity and concentration of an anti-thrombic agent at apredetermined temperature.
 81. The collagenous connective tissuespecimen recited in claim 80 wherein said polyglycol is selected fromthe group consisting of polyethylene glycol and polypropylene glycol.82. The collagenous connective tissue specimen recited in claim 80wherein said salt is selected from the group consisting of sodiumchloride, potassium chloride, sodium bromide, potassium bromide, sodiumsulfate, potassium sulfate, ammonium chloride and ammonium sulfate. 83.The collagenous connective tissue specimen recited in claim 80 whereinsaid oxidizing agent is hydrogen peroxide.
 84. The collagenousconnective tissue specimen recited in claim 80 wherein saidanti-inflammatory agent is selected from the group consisting ofindomethacin, ibuprofin, aspirin, choline salicylate, difunisal,magnesium salicylate, magnesium choline salicylate, salsalate,flurbiprofen, fenoprofen, ketoprofen, naprosen, naproxen sodium,oxaprozin, diclofenac sodium, diclofenac misoprostol, etodolac, indocin,ketorolac, natumetone, sulindac, tolmetin, sulfinpyrazone, dipyridamole,ticlopidine, valdecoxib, rofecoxib, piroxicam, meloxicam, meclofenamatesodium, mefenamic, cyclophosphamide, cyclosporine micromulsion,chlorambucil, anagrelide, clopidogrel, and cilostazol.
 85. Thecollagenous connective tissue specimen recited in claim 80 wherein saidanti-inflammatory agent in said third solution is an analgesic.
 86. Thecollagenous connective tissue specimen recited in claim 80 wherein saidanti-inflammatory agent in said third solution is an antipyretic. 87.The collagenous connective tissue specimen recited in claim 80 whereinthe temperature of said third solution is in the range of 4 to 25degrees Centigrade.
 88. The collagenous connective tissue specimenrecited in claim 80 wherein said anti-thrombic agent in said fourthsolution is selected from the group consisting of heparin, ardeparin,enoxaparin, tinzaparin, danapariod, lepiruden and hirudin.
 89. Thecollagenous connective tissue specimen recited in claim 80 wherein saidanti-thrombic agent is an anticoagulent.
 90. The collagenous connectivetissue specimen recited in claim 80 wherein said alcohol in said secondsolution is selected from the group consisting of ethanol, iso-propanol,and n-propanol.
 91. The collagenous connective tissue specimen recitedin claim 80 wherein the concentration of said alcohol in said secondsolution is in the range of 25% to 75%.
 92. The collagenous connectivetissue specimen recited in claim 80 wherein the concentration of saidanti-inflammatory agent in said third solution is in the range of 10 to200 mg/liter.
 93. The collagenous connective tissue specimen recited inclaim 80 wherein the concentration of said anti-thrombic agent in saidfourth solution is in the range of 100 to 1000 IU/ml.
 94. Thecollagenous connective tissue specimen recited in claim 80 wherein theconcentration of said polyglycol in said first solution is in the rangeof 1% to 15% and the molecular weight of said polyglycol is in the rangeof 2,000 Daltons to 20,000 Daltons.
 95. The collagenous connectivetissue specimen recited in claim 80 wherein the concentration of saidsalt in said first solution is in the range of 2.5M to 4.5M.
 96. Thecollagenous connective tissue specimen recited in claim 80 wherein theconcentration of said oxidizing agent in said first solution is in therange of 0.1% to 2%.
 97. The collagenous connective tissue specimenrecited in claim 80 wherein the temperature of said cold stabilizingsolution is in the range of 4 to 10 degrees Centigrade.
 98. Thecollagenous connective tissue specimen recited in claim 97 wherein saidcold stabilizing solution is a saline solution, further comprisingsoaking said collagenous connective tissue in said saline solution for aperiod less than 48 hours.
 99. The collagenous connective tissuespecimen recited in claim 97 wherein said cold stabilizing solution isan alcohol and water solution, further comprising soaking saidcollagenous connective tissue in said alcohol and water solution for aperiod less than 30 days.
 100. The collagenous connective tissuespecimen recited in claim 98 further comprising, after the step recitedin claim 19, placing said collagenous connective tissue in an alcoholsolution and soaking said collagenous connective tissue therein for aperiod less than 30 days.
 138. The collagenous connective tissuespecimen recited in claim 80 wherein said tissue is soaked in said firstsolution for a period in the range of 12 hours to 14 days.
 141. Thecollagenous connective tissue specimen of claim 80 where said phosphatebuffer is selected from the group consisting of sodium phosphate andpotassium phosphate.
 142. The collagenous connective tissue specimen ofclaim 80 wherein the concentration of said phosphate buffer is in therange of 0.02 to 0.1M with a pH in the range of 6.5 to 7.8.
 147. Thecollagenous connective tissue specimen recited in claim 80 furthercomprising the steps of soaking said collagenous connective tissue in a50′ ethanol solution containing predetermined quantities andconcentrations of glycerol, polyethylene glycol, indomethacin, andheparin, freeze-drying said collagenous connective tissue with alyophilyzer system, and sterilizing said collagenous connective tissuewith a hydrogen peroxide gas phase sterilizer.
 150. The collagenousconnective tissue specimen recited in claim 80 further comprising thesteps of freeze-drying said collagenous connective tissue with alyophilyzer system, and sterilizing said collagenous connective tissuewith a hydrogen peroxide gas phase sterilizer.
 153. The collagenousconnective tissue specimen recited in claim 80 wherein said oxidizingagent is ozone.
 156. The collagenous connective tissue specimen recitedin claim 153 wherein the range of concentration of said oxidizing agentis in the range of 1-500 ppm.